Tau-opathy model

ABSTRACT

The present invention relates to cell models of Alzheimer&#39;s disease. Transgenic yeast cells are described as models for the tau-opathy in Alzheimer&#39;s disease. These cell models comprise recombinant DNA constructs comprising control sequences and a cDNA sequence encoding a human tau-isoform and another similar construct comprising a protein kinase that is capable, directly or indirectly, of modulating the phosphorylation of the microtubule-associated protein tau. The transgenic cells are useful for high-throughput testing for potential therapeutic agents for Alzheimer&#39;s disease and other neurodegenerative disorders.

FIELD OF THE INVENTION

The present invention relates to a yeast model for the tau-opathy as inAlzheimer's disease and other neurodegenerative disorders. This model ofengineered yeast can be used in pharmaceutical screening and formodelling neurodegenerative diseases (e.g., Alzheimer's disease,frontotemporal dementia with Parkinsonism) and for in vivo modelling ofprotein tau biochemistry. It can be used as an assay, automated assay orhigh through put screening assay for identifying agents, compounds orchemical signals that directly or indirectly affect the biochemistry oftau (protein tau or tau-protein) and in particular of protein tauphosphorylation, comprising the steps of: growing the yeast cell line inappropriate media, said yeast cell comprising an introducedpolynucleotide or DNA sequence, an allelic variant, minigene or ahomologue thereof, that encodes for protein tau, protein tau isoforms orfunctional homologues thereof and expresses or overexpresses protein tauor functional homologues thereof and wherein said yeast cell comprisinga protein kinase that is capable directly or indirectly of modulating ofprotein tau, adding the test compound or chemical signal to the media;and measuring the extend to which the protein tau or functionalhomologues thereof are phosphorylated.

Alzheimer's disease, a neurodegenerative disease which is targeted byscreening assay of present invention, is the most common form of seniledementia, affecting approximately 5% of individuals over the age of 65and 20% of those over the age of 80. It has been estimated that thereare between 2.5 and 3 million patients suffering from Alzheimer'sdisease in the USA and up to 6 million in Europe. These figures willincrease exponentially over the next decades as the proportion ofelderly in the population increases exponentially and because theincidence of Alzheimer's disease itself increases exponentially withage.

To date, there are neither accurate methods for early diagnosis, nor anymethod or drug for the effective treatment of Alzheimer's disease. Thedevelopment to market of a therapeutic intervention for this major humandisease therefore represents a significant commercial opportunity.

BACKGROUND OF THE INVENTION

Alzheimer's disease is a neurodegenerative disorder characterisedhistopathologically by the loss of synapses on particular groups ofneurones and eventually in a later clinical stage, by the loss of theseneurones themselves. Post-mortem pathology reveals the abundant presenceof two principal lesions within the brain, named senile plaques andneurofibrillary tangles (Delacourte, 1999).

The mechanisms that cause the synaptic and neuronal loss are still notunderstood. Several different mutations in the gene coding for theamyloid precursor protein (APP) in some families with early onsetfamilial Alzheimer's disease (EOFAD) supports a “amyloid first”hypothesis in which the extracellular deposition of amyloid peptidesderived from APP is an early pathogenic event (for reviews see Hardy etal, 1998; Selkoe, 2000). In most other families with EOFAD, the diseaseis caused by mutations in the gene coding for Presenilin-1 (PS1), whilein some rare families affected members carry a mutant Presenilin-2 gene(PS2). Increased amyloidogenic APP metabolism and deposition of amyloidpeptides are regarded as a primary pathogenic event, however, which doesnot imply that a general consensus is reached on the primary pathogeniceffect of and how such deposition of amyloid peptides in the brain wouldresult in neurodegeneration and dementia.

Senile plaques are extracellular fibrillar deposits of amyloid peptides,surrounded by dystrophic neurites, which contain tau-aggregates. Amyloiddeposits are found in the brain parenchym but also prominently in thevascular walls of the deeper cerebral blood vessels in all cases ofAlzheimer's disease, be they early onset or late onset or sporadiccases. The amyloid deposits in senile plaques and in cerebral bloodvessels contain amyloid peptides of 40 and of 42 amino acids in length.These are derived by proteolytic cleavage from the larger amyloidprecursor protein (APP), a membrane-anchored glycoprotein, 100 to 110kDa in size.

On the other hand, neurofibrillary tangles (NFT) are intra-neuronalinclusions of paired helical filaments (PHF) which themselves areaggregates of protein tau, a microtubule-associated protein. Protein taurefers to a set of up to 6 isoforms of this cytosolic phosphoprotein(about 60-70 kDa). Hyper-phosphorylation of tau is thought to representthe principal cause of its aggregation into PHF and all subsequentmalfunctions in terms of cytoskeletal and synaptic stability, neuriteoutgrowth and axonal transport. Neurones containing neurofibrillarytangles are to be considered heavily compromised and not able tofunction normally. Neuronal death of such cells is evident from thepresence of “ghost” tangles in brain of Alzheimer's disease patients,i.e. residues of dead tangle-bearing neurones from which essentiallyonly the insoluble neurofibrillary tangles have been remained.

Alzheimer disease targets are currently studied by transgenic micemodels for Alzheimer's disease (for reviews see Van Leuven 2000;Dewachter et al., 2000). These models focus on APP and PS1, becausemutations in APP and PS1 cause EOFAD and because Down's patients(trisomy 21 on which the APP gene is located) develop classicalAlzheimer's disease pathology in their 2^(nd) to 3^(rd) decade, due tothe overexpression of APP by the gene-dosage effect. Single and doubletransgenic mice, i.e. APP and APP×PS1 transgenic mice in whom thetransgenes contain one or more EOFAD clinical mutations, show aprominent and robust amyloid pathology phenotype. This recapitulates theamyloid pathology of AD patients almost exactly in terms of deposits ofamyloid in the parenchym and in the cerebral blood vessels (Van Dorpe etal, 2000; Dewachter et al, 2000). Significantly, and very remarkably,although hyper-phosphorylation of tau is observed in the swollenneurites surrounding the neuritic plaques in these mice, noneurofibrillary tangles have been formed in the brain of theseamyloid-bearing transgenic mice (Van Dorpe et al, 2000).

It is a matter of strong debate if the cognitive deficits demonstratedin the transgenic APP and APP×PS1 mice, are relevant for the cognitivedeficits in the human Alzheimer patients. In brain of patients, thecognitive decline correlates much better with the presence and site ofdystrophic neurites containing PHF and neurofibrillary tangles than withamyloid plaque deposition. A popular hypothesis holds that even though aprimary pathogenic event must involve aberrant APP metabolism, themechanism by which the neurone degenerates is essentially a dysfunctionor disruption of the neuronal cytoskeleton. This impairs the axonal, andperhaps also dendritic transport, and is due to thehyper-phosphorylation of tau, which eventually also results in itsdeposition as PHF.

On the other hand, transgenic mice have been generated that overexpresshuman protein tau, either wild-type or a clinical mutant that give riseto another type of dementia, i.e. frontotemporal dementia withParkinsonism linked to chromosome 17 (FTDP-17) (for review see Heutink,2000).

Although these tau-transgenic mice do not develop an Alzheimer relatedphenotype, they demonstrate the massive and pathologic interference ofhuman tau with axonal transport, causing “ballooning” of the axons,axonal degeneration and as a secondary phenomenon, muscular wasting andmotoric problems(Spittaels et al, 1999, 2000). This phenotype is, mostamazingly, “rescued” by GSK-3β in double transgenic mice, in which nextto human tau also GSK-3β is expressed (Spittaels et al, 2000). In thedouble transgenic mice, the increased level of hyper-phosphorylation ofprotein tau correlates with decreased ballooning, alleviation of theaxonopathy and of the motoric and muscular problems (Spittaels et al,2000). This clearly indicates a duality in the role of GSK-31 and otherkinases that are eventually (or obligatorily) implicated in thesignalling pathways up- or down-stream of GSK-3β. These findingsevidently complicate any existing scheme in which hyper-phosphorylationof tau is suggested or advocated to be the direct cause of the axonalproblem. In this respect, the transgenic tau or GSK-3β mice are notsuitable as models to elucidate the molecular mechanisms by which thetau-pathology is “triggered” and “executed”. Such mechanisms must andcan be studied in cellular paradigms, preferable as simple as possiblein order to define the essential and fundamental features of signallingpathways and their phenotypic implications. Present invention disclosessuch cellular paradigm which besides ease of manipulation, bothgenetically and epi-gentically in terms of environmental and mediumparameters also provides a simple system which moreover is an essentialbonus for high-throughput screening purposes.

SUMMARY OF THE INVENTION

This invention discloses engineered yeast cells that are models foraspects of Alzheimer's disease and for another neurodegenerativedisease, such as frontotemporal dementia with Parkinsonism, in whichsaid engineered yeast cells express a human wild-type or mutant proteintau isoform, by introducing a DNA sequence encoding one of theseisoforms. A further aspect of the invention is that the engineered yeastcells express a human wild-type or mutant protein tau isoform, byintroducing a DNA sequence encoding one of these isoforms and are alsocapable of expressing a tau-kinase that is a direct or indirectmodulator of the phosphorylation state of the protein tau isoformexpressed in the same cell. In a further aspect of the invention saidengineered yeast cells contain introduced DNA sequences that encode andare capable of expressing the said protein tau and the said protein-taukinase that are capable, directly or indirectly, to modulate thephosphorylation state of the protein tau. In yet another aspect of theinvention said engineered yeast cells contain an introduced DNA sequenceencoding a control sequence, correctly integrated to allow theexpression of human protein tau, isoform protein tau or functionalhomologous thereof and also comprises a DNA sequence encoding andcapable of expressing a protein tau kinase or other kinase, capable ofdirect or indirect modulation of the phosphorylation state of proteintau

The DNA sequence of present invention encoding and capable of expressinga protein kinase that is capable, directly or indirectly, of modulatingthe phosphorylation-ste of protein tau can be either endogenous, but canor may also be introduced to establish or bring about increasedproduction of the chosen kinase, such as co-factored or accessorysubunits or activators or modulators.

The invention includes also the progeny and all subsequent generationsof the cells into which the said DNA sequence(s) were introduced.

The engineered yeast cells of present invention are used as a yeastmodel for a yeast model for the tau-opathy as in Alzheimer's disease andother neurodegenerative disorders. This model of engineered yeast can beused in pharmaceutical screening and for modelling neurodegenerativediseases (e.g., Alzheimer's disease, frontotemporal dementia withParkinsonism) and for in vivo modelling of protein tau biochemistry. Itcan be used as an assay, automated assay or high through put screeningassay for identifying agents, compounds or chemical signals thatdirectly or indirectly affect the biochemistry of tau (protein tau ortau-protein) and in particular of protein tau phosphorylation,comprising the steps of: growing the yeast cell line in appropriatemedia, said yeast cell comprising an introduced polynucleotide or DNAsequence, an allelic variant, minigene or a homologue thereof, thatencodes for protein tau, protein tau isoforms or functional homologuesthereof and expresses or overexpresses protein tau or functionalhomologues thereof and wherein said yeast cell comprising a proteinkinase that is capable directly or indirectly of modulating of proteintau, adding the test compound or chemical signal to the media; andmeasuring the extend to which the protein tau or functional homologuesthereof are phosphorylated.

ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The invention is based on the notion that modelling of allneuro-degenerative aspect of Alzheimer's disease is a most important andessential requirement.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1:

Heterologous protein tau expression in wild type S. cerevisiae. Westernblot analysis of crude cell extracts from different wild type strainsand isogenic mds1 deletion strains using the Tau-5 antibody to detectexpression of either human tau-wt or tau-P301L (Panel A) and GFP-tau-wtor GFP-tau-P301L fusion proteins (Panel B). Expression of GFP-fusionswas further analyzed by fluorescence microscopy (Panel B)

FIG. 2:

Phosphorylation mapping. The phosphorylation map assay was performed oncrude extracts derived from the wild type strain, the isogenic mds1deletion strain and the isogenic pho85 deletion strain that expresseither human tau-wt or tau-P301L. Panel A: strains from the W303-1Agenetic background. Panel B: strains from the BY4741 genetic background.

Different antibodies were used as indicated; Tau-5 asphosphorylation-independent “pan”-tau antibody to visualize the totalamount of tau-protein blotted, AT-8 and AD2 as antibodies that recognizedifferent phosphorylated tau-epitopes, and Tau-1 as antibody thatrecognizes non-phosphorylated tau-epitopes.

FIG. 3:

Effect of PP2A overexpression on tau-phosphorylation. Western blotanalysis of the W303-1A wild type strain or the same strainoverexpressing the protein phosphatase Pph21. Immunodetection wasperformed using the phosphorylation independent antibody Tau-5 or theantibody that recognizes non-phosphorylated epitopes Tau-1 as indicated.

FIG. 4:

GSK3 complementation analysis. The human GSK3-beta was expressed in ayeast mds1 deletion strain to demonstrate functional complementation fortau-phosphorylation Phosphorylation of tau-wt and tau-P301 L wasmonitored in the W303-1A wild type, the mds1 deletion strain and themds1 deletion strain expressing human GSK3-beta. Western blot analysiswas performed on crude cell extracts using the AD2 antibody for whichimmunoreactivity is dependent on phosphorylation of the Ser-396 andSer-404 epitopes. Immunodetection with Tau-5 served as control.

FIG. 5:

Validation of the yeast model to study signal transduction cascades withtau-phosphorylation as marker. Phosphorylation of heterologous expressedhuman protein tau-wt and tau-P301L was monitored in different strainsdeficient for kinases previously demonstrated to operate ininter-connecting signal transduction cascades in S. cerevisiae. Theupper panels show phosphorylation mapping in the W303-1A sch9 deletionstrain, the middle panels for the W303-1A yak1 deletion strain and thelower panels for strain deleted for the different Tpk subunits of PKA.Corresponding human homologues kinase activities are indicated betweenbrackets. Immunodetection was performed using the antibodies AD2, Tau-5and Tau-1.

FIG. 6:

Effect of heterologous expression of human protein tau on physiologicalprocesses in yeast. Panel A: The effect on tau expression on pseudohypaldifferentation upon nitrogen limitation was monitored in the 1278strain. As a control for full pseudohyphal differentiation, the straintransformed with the empty plasmid is included in the picture on theleft. The inhibitory effect of tau-P301L expression or tau-wt expressionare visualised respectively in the middle and the right picture. Panel Band C: The effect of expression of tau-wt and tau-P301L on benomylsensitivity of the BY4741 wild type strain (panel B), the isogenic mds1deletion strain (panel B) and the isogenic pho85 deletions strain (B andC). Panel B shows the growth curves (OD600 nm) in liquid cultures in thepresence of 40 microgram benomyl as measured with the BIOSCREEN Cworkstation. Panel C shows the plate assay for growth of serialdilutions of the pho85 deletion strains transformed with, from left toright, the empty plasmid, tau-P301L or tau-wt. The final benomylconcentrations in the plates are as indicated

FIG. 7:

Solubility assay of heterologous expressed human protein tau in S.cerevisiae. Sequentially obtained protein extracts obtained from thewild type BY4741 strain and the isogenic mds1 deletion strain expressingtau-wt or the pho85 deletion strain expressing either tau-wt ortau-P3041L , as indicated, were analysed by western blot analysis usingthe Tau-5 antibody. The lanes are indicated and contain samples of thesupernatant or the pellet obtained from subsequent extraction in highsalt reassembly buffer (RAB), detergent containingradioimmunoprecipitation assay buffer (RIPA) or 70% formic acid (FA)

TERMINOLOGY AND DEFINITIONS

For purposes of the present invention, the following terms are definedbelow.

The term “cell lines” is used in the present invention refers toeukaryotic cell lines preferably in the form of a cell line that issuitable for continuos culture, either in suspension or attached on asuitable carrier and is a recombinant cell that may be obtained bymanipulation of cells using conventional techniques of recombinant DNAtechnology.

The term “yeast cell” herein is used to mention single-celled fungi ofthe phylum Ascomycota that reproduce by fission or budding and arecapable of fermenting carbohydrates into alcohol and carbon dioxide.Yeast cells of the species Saccharomyces cerevisae are preferred formanipulation to incorporate DNA sequences in accordance with the presentinvention. Such cells do not normally express a protein tau but arecapable of expressing human protein tau by introduction of a DNAsequence encoding human tau under the control of appropriate regulatoryDNA sequences. The resulting DNA sequence is considered a “recombinant”DNA sequence or a “transgene”.

The term “engineered yeast” is used herein to mention yeast cells,having a transgene or non-endogenous (i.e. heterologous) nucleic acidsequence present as a extrachromosomal element in stably integrated intoits germ line DNA (i.e. in the genomic DNA). Heterologous nucleic acidis introduced into the germ line of such engineered by geneticmanipulation

The term “introduced DNA sequence” is used herein to denote a DNAsequence that has been introduced into a cell and which may or may notbe incorporated into the genome. The DNA sequence may be a sequence thatis not endogenous to the chosen type of cell, that is endogenous but isnot normally expressed by that cell or that is endogenous and isnormally expressed but of which over-expression is desired. The DNAsequence may be introduced by any suitable transfection techniqueincluding electroporation, calcium phosphate precipitation, lipofectionor other known to those skilled in the art. The sequence may have beenintroduced directly into the cell or may have been introduced into anearlier generation of the cell.

The term “Transgene” means any piece of DNA which can be inserted into acell, and preferably becomes part of the genome of the resultingorganism (i.e. either stably integrated or as a stable extrachromosomalelement). Such a transgene includes genes which are partly or entirelyheterologous (i.e. foreign) as well as genes homologous to endogenousgenes of the organism. Including within this definition is a transgenecreated by providing an RNA sequence with is reverse transcribed intoDNA and then incorporated into the genome, or an antisense agent ormolecule.

The terms “tau”, “protein tau” or “tau-protein” refer to a specific(poly)peptide or protein associated with the assembly, stability, orenhancement of the polymerisation of microtubules. Tau protein exists inup to 6 different isoforms in adult brain, while only 1 isoform isexpressed in fetal brain, but all are generated from a single gene onhuman chromosome 17 by alternative mRNA splicing. The most strikingfeature of tau protein, as deduced from molecular cloning, is a stretchof 31 or 32 amino acids, occurring in the carboxy-terminal part of themolecule, which can be repeated 3 or 4 times. Additional diversity isgenerated through 29 or 58 amino acid-long insertions in the N-terminalpart of tau molecules ( Billingsley and Kincaid, 1997).

Under normal circumstances protein tau promotes microtubule assembly andstability in the axonal compartment of neurones. The microtubule-bindingdomain in protein tau is localised in the repeat region of tau (255-381)and is modulated by adjacent regions: the carboxyterminal tail (382-414)and the proline-rich region (143-254). Stability and bundling of themicrotubules is mediated by a short hydrophobic zipper in thecarboxyterminal tail of tau Both assembly and stability are regulated byalternative mRNA splicing and phosphorylation.

In normal adult brain, protein tau contains 2 to 3 mol phosphate permole of protein tau while phosphorylation of different sites in normaltau follows different developmental profiles (Brion, 1998). Abnormalprotein tau variants of 60, 64 and 68 kDa have been detected exclusivelyin brain areas showing neurofibrillary changes and senile plaques(Delacourte et al, 1999). The abnormal electrophoretical behaviour oftau is due to phosphorylation since alkaline phosphatase treatment ofthese tau molecules changes their molecular mass to that of normal tau.Currently abnormal phosphorylation sites have been detected in PHF-tauat positions 46, 231, 235, 263 and 396. In four of these sites, thephosphorylated residue is followed by a proline residue, indicating thata s proline-directed kinase Is involved In some of the abnormalphosphorylations of tau. In addition to these sites ten others arepresent in htau40, two of which are also abnormally phosphorylated, asindicated by antibody reactivity.

Detection of PHF-tau in brain extracts is either via antibodies or viachanges in molecular weight or electrophoretic mobility. The abnormalphosphorylation of tau in Alzheimer's disease is due to a shift in thephosphatase/kinase equilibrium. In vitro several kinases canphosphorylate tau: cdc2 and cdk5-kinases, MAP kinases, glycogen synthasekinases (GSK3) among others. Phosphatases are less well studied in brainin general and hardly in Alzheimer's disease, and only phosphatase 2Ahas been shown to be capable in vitro to dephosphorylate the abnormallyphosphorylated sites I tau.

The term “tau gene” means a tau gene an allelic variant, minigene, ahomologue thereof or a gene, that encode for htau40, for protein tau, anisoform of tau or functional homologues thereof or at least a portionthereof

As used herein, the term “minigene” refers to a heterologous geneconstruct wherein one or more nonessential segments of a gene aredeleted with respect to the naturally-occurring gene. Typically, deletedsegments are intronic sequences of at least about 100 basepairs toseveral kilobases, and may span up to several tens of kilobases or more.Isolation and manipulation of large (i.e., greater than about 50kilobases) targeting constructs is frequently difficult and may reducethe efficiency of transferring the targeting construct into a host cell.Thus, it is frequently desirable to reduce the size of a targetingconstruct by deleting one or more nonessential portions of the gene.Typically, intronic sequences that do not encompass essential regulatoryelements may be deleted. Frequently, if convenient restriction sitesbound a nonessential intronic sequence of a cloned gene sequence, adeletion of the intronic sequence may be produced by: (1) digesting thecloned DNA with the appropriate restriction enzymes, (2) separating therestriction fragments (e.g., by electrophoresis), (3) isolating therestriction fragments encompassing the essential exons and regulatoryelements, and (4) ligating the isolated restriction fragments to form aminigene wherein the exons are in the same linear order as is present inthe germline copy of the naturally-occurring gene. Alternate methods forproducing a minigene will be apparent to those of skill in the art(e.g., ligation of partial genomic clones, which encompass essentialexons but which lack portions of intronic sequence). Most typically, thegene segments comprising a minigene will be arranged in the same linearorder as is present in the germline gene, however, this will not alwaysbe the case. Some desired regulatory elements (e.g., enhancers,silencers) may be relatively position-insensitive, so that theregulatory element will function correctly even if positioneddifferently in a minigene than in the corresponding germline gene. Forexample, an enhancer may be located at a different distance from apromoter, in a different orientation, and/or in a different linearorder. For example, an enhancer that is located 3′ to a promoter ingermline configuration might be located 5′ to the promoter in aminigene. Similarly, some genes may have exons, which are alternativelyspliced, at the RNA level, and thus a minigene may have fewer exonsand/or exons in a different linear order than the corresponding germlinegene and still encode a functional gene product. A cDNA encoding a geneproduct may also be used to construct a minigene. However, since it isoften desirable that the heterologous minigene be expressed similarly tothe cognate naturally-occurring non-human gene, transcription of a cDNAminigene typically is driven by a linked gene promoter and enhancer fromthe naturally-occurring gene. Frequently, such minigene may comprise atranscriptional regulatory sequence (e.g., promoter and/or enhancer)that confers neuron-specific or CNS-specific transcription of theminigene alpha-synuclein encoding sequences.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues.

As used herein, “isoform tau”, “tau isoform”, “isoform protein tau”,“protein tau isoform” refer to a (poly)peptide or protein that isencoded by at least one exon of the tau gene

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves engineered yeast cell-lines that expressprotein tau, preferably human tau protein, or functional isoforms orhomologues thereof, to the proteins produced by these cell-lines, to thephosphorylation pattern of tau In these cell-lines and to theirapplications and further it discloses a process for screening for drugsfor the therapy of neurodegenerative disease or more particular ofdementia of the Alzheimer type or of the frontotemporal dementia withParkinsonism and for diagnosis of this dementia by drugs thatspecifically bind to such tau-proteins or that affect tau proteinphosphorylation. It thus provides solution for a long felt need. Ofthese adult-onset dementia, Alzheimer's disease (AD) is the most commonform. At present, no reliable biochemical test is available forantemortem diagnosis of AD. The disease is therefore diagnosedclinically on the basis of exclusion of other forms of dementia. Thediagnosis can be confirmed neuropathologically by the demonstration oflarge amounts of neuritic (senile) plaques and neurofibrillary tangles(NFT) in particular brain regions, i.e. hippocampus, subiculum andenthorrhinal cortex.

Neurofibrillary tangles consist of paired helical filaments (PHF) andmicrotubule-associated protein tau is the major protein component of PHFand thus of NFT (Brion., 1998; Delacourte, 1999. The microtubuli-bindingdomain of tau is tightly associated with the core of PHF while taupeptides may represent only a small portion of the major component ofPHF.

The present invention also concerns different modified yeast cells thatexpress or lack different isoforms or mutant forms of human protein tau,in combination with expression or deficiency of human and yeast kinases,to allow the reliable and specific production of specified,phosphorylated isoforms of protein tau, as present in brain andcerebrospinal fluid of Alzheimer patients.

The invention also concerns the specified recombinant phosphoproteinsproduced in the above-said modified yeast cell-lines.

The invention furthermore concerns the phospho-epitopes of tau proteinas present in brain homogenates or in body fluids such as cerebrospinalfluid, which are recognised by specified monoclonal antibodies, in useor to be produced, in the field of Alzheimer's disease.

Furthermore, the invention concerns standardised products in the form ofisoforms of protein tau that are essentially useful for the detectionand diagnosis in vitro of Alzheimer's disease, other dementia's, otherthau-opathies or other brain diseases, all involving abnormalphosphorylation of tau proteins.

Furthermore, the novel engineered yeast cell-lines of present inventionare characterised in that they specifically produce naturally occurringabnormally phosphorylated tau of which the phosphorylation state isconfined to a particular specified region of the tau molecules, orrecombinant non-phosphorylated tau which by treatment withproline-directed kinases can provoke the phosphorylation of, amongstothers, Ser-Pro or Thr-Pro sites as specified. Proline-directed kinasessuch as MAP kinases , cdc2 kinases, glycogen synthase kinases and cdk5kinases can be purified from various sources, such as geneticallyengineered yeast strains or can be present in brain extracts. Thephosphorylation of tau by these kinases is abolished or greatlydiminished when one or more of the following serines/threonines aremutated to an amino acid such as Ala: T153, T175, T181. S199, S202,T205, T212, T217, T231, S235(Billingsley and Kincaid, 1997).

Consequently, the structure-function relation of said phosphorylatedmutant tau proteins can be characterised, in addition to those ofnon-phosphorylated tau protein as produced in specified kinase deficientyeast cell-lines also the subject of this invention, and said yeastcell-lines will be used to define in vivo the effect on a number ofspecified biological processes, such as formation of mitotic bundles, ofpseudo-hyphen, of scar-sites, of cell-size, of cell-growth in definedconditions, of response to external signals, agent or compound. Thebiological processes are also the subject of the present invention andare defined in the engineered yeast cell-lines in which the productionof specified human tau proteins and of specified human protein kinasesresults in abnormal phosphorylation of protein tau and of itsinterference with micro-tubular structures and with the resultingabnormal transport and functions mediated by protein tau, as specifiedabove, to finally result in abnormal fibrillar inclusions andaggregates, characterised by phospho-epitopes referred to as a “PHF-tauepitopes” as in the brain of patients suffering from Alzheimer's diseaseand other tau-opathies and dementia's (Heutink, 2000).

The expression “specified abnormally phosphorylated tau protein”corresponds to the fact that the engineered yeast cell-lines of theinvention CAN produce abnormally phosphorylated tau isoforms that aredefined by specified compounds such as, but not restricted to,monoclonal antibodies, without reacting or cross-reacting with normaltau proteins as present in cells, in brain or in CSF.

The expression “form a specific complex” means that the phosphorylatedprotein tau isoforms of the invention in the engineered yeast strains asthe biological system of the invention, presents as higher-molecularweight products under conditions as described or mentioned in one of thefollowing techniques:

A very possible factor that could be involved in Alzheimer's disease ishyper-phosphorylation of tau. This can be caused by disturbedequilibrium activity of kinases and Phosphatases, which regulate thephosphorylation status of tau. Kinases involved directly inphosphorylation of tau have been identified in vitro by incubation ofcandidate kinases with recombinant tau produced in bacteria, and haverevealed mitogen activated kinases (MAP-kinases) and glycogen synthasekinase-3β (GSK-3β), cdk5 kinase with any of his activating subunits(p70, p39, p35, p29, p25, . . . ) and other unidentifiedproline-directed kinases to be capable to phosphorylate tau on epitopesas encountered in PHF-tau (for reviews see Billingsley and Kincaid,1997, Mandelkow and Mandelkow, 1998; Delacourte, 1999 ). These areevidently, but not the only candidate kinases that phosphorylate tau invivo.

These kinases usually are involved in signalling pathways and requireactivation by some other mechanism, generally also phophorylation orde-phosphorylation, but eventually proteolytic cleavage.

The mechanisms by which GSK-3β activity is controlled is byphosphorylation on tyrosine, serine and/or threonine residues by proteinkinase C (PKC). MAP kinases are activated by phosphorylation on tyrosineand threonine residues by a MAP kinase MEK) which is self phosphorylatedon serine and threonine residues by MEK kinase(s), possibly identical orrelated to the proto-oncogenes cRaf-1 or mos, or to the genes SteII andByr2. These kinases can then be involved indirectly in controllingphosphorylation of tau and thereby used in the present invention toidentify candidate kinases and pathways.

Modelling of Alzheimer's disease PHF-type hyperphosphorylation of taumay therefore be achieved by genetic manipulations using those kinasesinvolved directly or indirectly in the phosphorylation of tau. Theinventors observed that the deletion or introduction of GSK-3β from orinto cultured yeast cells that were engineered and capable to expresshuman tau, was particularly effective in achieving hyper-phosphorylationof tau as in brain of Alzheimer patients and in brain of doubletransgenic mice (tau x GSK-3β).

Accordingly, in an embodiment of the present invention, the kinase thatis directly or indirectly modulating the phosphorylation of themicrotubule-forming protein tau is preferably glycogen synthasekinase-3β.

Methods for screening potential therapeutic agents using cell lines arevery well established and well known to those skilled in the art, ascell-based models are generally used as most early stage screeningmethods involving immunoassays or cell-morphological characteristics. Inan embodiment of present invention example antibodies, for example,monoclonal antibodies, may be used against phophorylated andnon-phosphorylated tau epitopes for which many examples are known tothose skilled in the art. For example, antibodies Tau-1 and tau-5, aremouse monoclonal antibodies that react with normal tau but not PHF-tau,while AT8, AT100, AT180, AT270, PHF1, MC1, SMI31, SMI34, SMI310, ALZ-50,among others are mouse monoclonal antibodies that recognise PHF-tau butdo not react with normal tau in normal human brain.

Screening may be carried out as follows: Recombinant cells of thepresent invention are incubated with a potential therapeutic factor fora specified time in specified conditions, and the cells are thenincubated with specified antibodies after which the extent of binding ismeasured as an index of the hyper-phosphorylation status of tau.Effective candidate drugs will decrease hyperphosphorylation of tau.Preferably, the screening assay is performed by ELISA since these can beperformed rapidly and on a large scale using microtitre plates and(semi-)automated apparatus.

Accordingly, the invention also provides a screening assay kitcomprising:

-   -   i) engineered yeast cells    -   ii) an antibody, preferably a monoclonal antibody, capable of        binding selectively to phoshorylated tau. Alternatively the        present invention also provides a screening assay further        comprising    -   iii) an antibody, preferably a monoclonal antibody, capable of        binding selectively to un-rphoshorylated (normal) tau.

Advantageously the screening assay is an enzyme-linked immunosorbentassay.

The recombinant cell lines of the present invention may also serve asmodels for other neurodegenerative disorders in which there is evidenceof abnormal cytoskeletal protein phosphorylation; these conditionsinclude frontotemporal dementia, vascular delentia, Parkinson's disease,amyotrophic lateral sclerosis, senile dementia of the Lewy body type(SDLT), and stroke.

Accordingly, the recombinant cell lines of the present invention may beused for the study of such conditions and screening of therapeuticagents for such conditions.

The invention uses introduction and expression of tau and tau-kinases incells that do not normally express tau, i.e. yeast cells. The cells aremanipulated to express tau by the introduction of its cDNA under thecontrol of a specified controlling promoter sequence, generally using acloning vector, for example, a plasmid. These methods and procedures arewell-known to those skilled in the art.

Human GSK-3β cDNA is preferably used in the production of constructs, asa modification of the wild type gene. For this, site-directedmutagenesis was used to substitute the serine residue at codon 9 by analanine residue in GSK-3β, to prevent own-regulation of its activity byphosphorylation at serine 9, which is known to those in he field, to bea normal inactivation signal.

EXAMPLES General Methodologies used in the Invention

Yeast deletion strains: Genomic deletions of specific genes were made inthe S. cerevisiae W303-1A strain (Thomas, B. J. and Rothstein, R. J.,Cell 1989; 56: 619-630), the BY4741 strain (Brachmann et al., Yeast1998; 14: 115-132) or the Σ1278b strain (Kron, S. J. Trends Microbiol.1997;5:450-454) as indicated. They were obtained by PCR product-directedgene disruption as described previously (Brachmann et al., Yeast 1998;14: 115-132) using oligonucleotides listed in Table 1 and using the pRSvectors as templates for auxotrophic selectable markers. Deletions werechecked by Southern Blot analysis (Sambrook et al. Molecular Cloning, aLaboratory Manual, 2^(nd) edn. 1989; Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press) or by PCR analysis. The PKA deletionstrains ASY62 and ASY63 were kindly provided by S. Garrett and have beendescribed previously (Smith A. et al., EMBO J., 1998; 17:3556-3564)

cDNA expression constructs: Wild-type human tau and mutant tau-P301Lwere transformed in yeast as recombinant constructs that contained thetriose phosphate isomerase (TPI) promoter (Alber T, Kawasaki G., J MolAppl Genet 1982;1:419-434), allowing the tau-cDNAs to be constitutivelyexpressed. For this purpose, the tau-wt 2N/4R cDNA, the tau-P301L 2N/4RcDNA or the corresponding GFP cassettes containing the tau cDNAs fusedin frame to the coding sequence of GFP, were ligated into the EcoRI-XhoIsites of the yeast/E. coli shuttle vector pJW212 which is a derivativeof pYX212 (R&D systems Europe Ltd., Abingdon, UK). The cloning of thecDNA inserts was confirmed by sequence analysis using a method based onthe standard dideoxy sequence analysis (Sanger et al., Proc. Natl. Acad.Sci. USA 1977;74: 5463-5467). The resulting tau-expression plasmids weretransformed into the appropriate yeast strains according to the protocoloutlined by Gietz R. D. and Schiestl R. H. (Methods in Molecular andCellular Biology 1995;5: 255-269). Transformed cells were plated onselective glucose-containing medium without uracil (SD-ura) as specifiedby Sherman et al. (Methods in Yeast Genetics. 1986; Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press). The human GSK3p cDNAexpression vector was derived from pKT10-GSK3β (Andoh et al., Mol. Cell.Biol. 2000; 20:6712-6720). This plasmid was cut with BsmI, blunt endedwith T4 DNA polymerase and ligated with a PvulI-EcoRV fragmentcontaining the KanMX gene from pUG6 (Guldener U. et al., Nucleic AcidsRes. 1996; 24:2519-2524). To select the yeast transformants that containthis huGSK3β cDNA expression plasmid, cells were plated on YPD platessupplemented with kanamycin at a final concentration of 150 μg per ml.

Overexpression of PP2A: The PP2A overexpression plasmid contains theyeast PPH21 gene fused to the PGK promoter and the PGK terminator inpGKJW2. For this construction, the PPH21 gene was amplified with primersthat introduced a SmaI at the 5′ end and a BamHI at the 3′ end of thecoding sequence. The PCR product was then digested with SmaI and BamHI,purified and ligated into the SmaI and BglII restriction sites ofpGKJW2. The plasmid pGKJW2 was obtained by cloning a 1.8 kb HindIIIfragment of pMA91 that contains the PGK promoter, a BglII cloning siteand the PGK terminator (Mellor et al., Gene 1983; 24:1-14) into theHindIII site of YEpLAC181 (Gietz R. D. and Sugino A., gene1988;74:527-534). The original pUC19 multiple cloning site of YEpLAC181were then removed by an EcoRI-SalI double digest followed by T4polymerase treatment and ligation. Subsequently, a new multiple cloningcassette containing the restriction sites for BamHI, SmaI, EcoRI andBglII was introduced between the PGK promoter and the terminator byinserting a synthetic linker into the BglII site. Since pGKJW2 containsthe LEU2 gene, transformants were selected on minimal glucose-containingmedium without leucine (SD-leu).

Yeast culture: Yeast cells were cultured in YEP medium (2% (w/v) bactopeptone, 2% (w/v) yeast extract) or in the appropriate selective mediumin order to maintain plasmids in transformed strains. Media weresupplemented with 4% (w/v) glucose (YPD or SD) unless otherwisespecified. For pseudohyphal growth, cells were plated on nitrogenlimitation medium (1 mM asparagine, 0.17% (w/v) yeast nitrogen basewithout amino acids and without NH4SO4, 2% (w/v) glucose and 1.5% (w/v)agar). Cells were grown at 30° C. or 25° C. for different time periodsas specified.

Benomyl resistance assays: For benomyl (Methyl1-butylcarbamoyl-2-benzimidazolecarbamate) resistance assays, cells weregrown in YPD supplemented with 5 to 60 μg benomoyl as indicated. Growthwas monitored either by plating serial dilutions or by measuring OD600in a Bioscreen C Microbiology Workstation (Thermo Labsystems, Helsinki,Finland) according to the manufacturers specifications.

Preparation of crude extracts for Western blotting: Yeast cells wereinoculated at a density of OD600 of 0.2 in 5 ml selective medium andgrown for 16 hours at 30° C. One milliliter of the culture wastransferred to a microcentrifuge tube and chilled on ice. The cells wererapidly harvested by centrifugation in a cooled (4° C.) microcentrifugeat maximal speed for 15 s and the pellet was resuspended in 50 μlprewarmed (95° C.) standard SDS-PAGE sample buffer. The mixture wasboiled for 15 min in order to denature and inactivate all proteinasesand phosphatases and then processed by Western blot analysis asdescribed below.

Tau-solubility tests: The solubility of protein tau was determined bysequential extraction of yeast cells (grown in SD-ura till an OD600 of2) with high salt reassembly buffer (RAB:1M Sucrose, 0.1M MES pH7.0, 1mM EGTA, 0.56 mM MgSO4) supplemented with a protease inhibitor mix(COMPLETE, Roche Diagnostics GmbH), detergent containingradioimmunoprecipitation assay buffer (RIPA:50 mM Tris pH8.0, 150 mNMNaCl, 5 mM EDTA, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1%(v/v) nonidet P40) and 70% formic acid as previously described (IshiharaT. et al., Neuron, 1999;24:751-762) Yeast cells were broken inreassembly buffer with glass beads by rigorous vortexing and the cellsuspensions were centrifuged at 50.000×g. Samples of both thesupernatants and the pellets were taken after each consecutiveextraction and stored for quantitative Western blot analysis using Tau-5antibodies.

Western Blotting: The denatured and reduced protein mixtures wereseparated by SDS-PAGE as performed under reducing conditions on either4-20% linear gradient gels or on 8% or 12% homogenous gels (Novex, SanDiego, Calif.). After electrophoresis, the proteins areelectrophoretically transferred to nitro-ellulose filters (Hybond-C,Amersham, UK) or to PVDF filters (ABI, San Fransisco, Calif.). Thefilters are blocked by incubation for 1 hour in PBS with 0.05%(v/v)Tween 20 and 5% (w/v) skimmed dried milk (blocking buffer). Thefilters are then incubated overnight with a specified monoclonalantibody or a specified polyclonal antiserum appropriately diluted insame blocking buffer. The filters are then washed three times inTween-PBS and treated for 1.5 h at room temperature with horseradishperoxidase-labelled rabbit anti-mouse IgG (Dakopatts, Denmark) diluted1/3000 in blocking buffer. After three washes in Tween-PBS,streptavidine-biotinylated horseradish peroxidase complex (Amersham),diluted 1/250 in blocking buffer, is applied for 1.5 h at roomtemperature. Thereafter, the filters are washed three times in Tween-PBSand once in PBS. The filters are then incubated in PBS containing 0.05%(w/v) diaminobenzidine and 0.03% (v/v) hydrogen peroxide untilbackground staining develops.

It should be clear that the formation of an Immunological complexbetween the monoclonal antibodies and the antigen is not limited to theprecise conditions described above, but that all techniques that respectthe immunochemical properties of the antibody and antigen binding willproduce similar formation of an immunological complex.

The phospho-epitopes are from 4 to 8 amino acids long and according tothis embodiment of the invention, can also be defined chemically insolution or in solid phase according to any of the techniques well knownin the art and compared to phosphorylated peptides as prepared accordingto techniques also known in the art. The detection of theimmunologically bound monoclonal antibody can be achieved byconventional technology known and comprised in the art, with a secondantibody that itself carries a marker or a chemical or physical group asa marker.

Monoclonal antibodies: We used the monoclonal antibody Tau-5(Pharmingen, San Diego, Calif.) that is directed against an epitope onhuman protein that is constitutively expressed on all human tau isoformsand independent of phosphorylation (“pan”-tau antibody). Monoclonalantibody AT-120 (Innogenetics, Ghent, Belgium) recognises a specialepitope, which is situated immediately C-terminally of T-231. Thereaction with protein tau is not phosphorylation-dependent in westernblotting, while in immunohistochemistry on human brain AT-120 reactsonly with the paired helical filaments (PHF) in the neurofibrillarytangles (NFT) in AD brain. The monoclonal antibodies AT-8 (Innogenetics,Ghent, Belgium) and AD-2 (gift from B. Pau, Lille, France) were used todetect specified epitopes on protein tau when phosphorylated.

The epitopes of these monoclonal comprise Ser(P)-1 99 and Ser(P)-202 forAT8 and Ser(P)-396 and Ser(P)404 for AD2. Similarly, the monoclonalantibody AT180 (Innogenetics, Ghent, Belgium) recognizes phosphorylatedprotein tau at Thr-181 and Thr-231.In addition, monoclonal antibodyTau-1 (Roche Molecular Biochemicals, Mannheim, Germany) was used as itsspecificity is complementary to that of AT8, i.e. Tau-1 recognises thesame epitope comprising Ser-199 and Ser-202 but only when these residuesare not phosphorylated.

Microscopy: Yeast cells grown on pseudohyphae-inducing nitrogenlimitation medium were scraped from plate, mixed with water and mountedon glass slides. GFP-transformed cells were grown till an OD600 of 3 inSD-ura and 3 μl culture were spotted on glass slides. Images wereprocessed with a ZEISS-axioplan laser microscope under a 100×oil-immersion objective.

Example 1 Expression of Human Protein Tau in Saccharomyces cerevisiae:Human Protein tau is Phosphorylated when Expressed in Yeast

To demonstrate that human protein tau, both wild-type and mutant P301L,can be expressed in yeast, we performed Western blotting on crudeextracts from transformed W303-1A and BY4741 wild-type strains andisogenic mds1 strains that lack one of the yeast genes encoding a kinasehomologous to the human tau-kinase GSK-3β. For detection we used themonoclonal antibodies Tau-5, which is the phosphorylation-indenpendent“pan”-tau antibody. The cell lines W303-1A -HuTau-wt andW303-1A-mds1-HuTau-wt have been patent deposited (IDA deposit) at theBelgian Coordiated Collection of Microorganisms—BCCM IHEM-Collection andreceived the numbers of respectively, IHEM 19160 and IHEM 1961.

Results: As shown in FIG. 1, we obtained considerable expression ofhuman wild-type tau and human mutant tau-P301L in yeast strains, wherebyits immuno-reactivity is retained as in human brain or in transgenicmouse brain, or in mammalian expression systems. This result holds truenot only in the wild-type yeast strains but also in the mds1Δ deletionstrains. In addition, the electrophoretic mobility of theimmuno-reactive wild-type tau proteins is slower in the wild typestrains when compared to the mds1Δ strains which is indicative for adifference in phosphorylation. This difference in mobility between thestrains is not obvious when mutant tau-P301L is expressed suggestingthat the P301L mutation in protein tau may interfere with itsphosphorylation.

Furthermore, as illustrated in FIG. 1B human protein tau can also beexpressed as a functional GFP-fusion product without loss ofimmunoreactivity.

The result of present invention demonstrate the surprising finding thatin a microbial eukaroytic cellular system the effect of mutations of tauon its phosphorylation can be analysed. The combination of thesesurprising findings constitute the needed proof-of-principle thatvalidates the entire experimental approach and strategy to analyseprotein tau, its phosphorylation and the consequences of mutations, in aless complex cellular system that is easily genetically modifiable. Itis the object of present invention to provide a completely novelexperimental system to address the importance of tau-mutations and thesignal-transduction cascades that are responsible for and lead tohyper-phosphorylation of protein tau.

Example 2 Expression of Human Protein Tau in Saccharomyces cerevisiae:Human Protein Tau is Phosphorylated in Yeast at the samePhospho-epitopes as in Brain of Alzheimer Patients

To confirm if the heterologous expressed protein tau ispost-translationally modified by phosphorylation in yeast, a moreelaborated phosphorylation mapping study was performed on crude extractsobtained from the transformed W303-1A and BY4741wild type strains andtheir isogenic mds1Δ (lacking one of the yeast genes encoding a kinasehomologous to the human tau-kinase GSK-3β) and pho85Δ strains (thelatter being deficient for a kinase which is homologous to the secondknown tau-kinase, i.e. cdk5). As monoclonal antibodies we made use ofAT-8 and AD2, which recognize only phosphorylated epitopes, and Tau-1,which detect non-phoshorylated epitopes

Results: The positive immunoreaction with all the different antibodieswas evident in all experiments performed. Some differences in intensityin the different combinations indicated a different extend ofphosphorylation as illustrated in the representative western blots shown(FIG. 2).

The reactivity with AT-8 is evident when wild-type or mutant human tauis expressed, and in addition appeared independent of the geneticbackground of the yeast strain (FIG. 2). In combination with the weakimmunoreactivity of monoclonal antibody Tau-1, recognising the sameepitope when not phosphorylated, these results indicate that thepathway(s) leading to the phospho-epitope defined by antibody AT8 isunder-active or under-represented in the yeast strains studied here.This opens various interesting possibilities for different experimentalapproaches, i.e. genetic complementation, external variables, cultureconditions, . . . to establish these pathway(s) and define them inmolecular terms.

Comparison of the immunoreactivity with antibody AT-180 in the wild typeand msd1Δ strain demonstrates the involvement of Msd1, c.q. one of thehuman GSK-3β homologues in yeast, in the control of phosphorylation ofThreonine-231, the phosphorylated residue that is comprised in thephospho-epitope defined by AT-180 (data not shown).

The most conspicuous result was the reaction of monoclonal antibody AD-2that was absent in the mds1Δ strain but not in the pho85Δ strain, c.q.the human cdk5 homologue (FIG. 2). This result demonstrated that theyeast Mds1 kinase is directly responsible for phosphorylation ofresidues Serine-396 and Serine-404 in human protein tau. This is anexcellent corroboration of our findings in the brain of GSK-3βtransgenic mice, in which the AD-2 epitope was implicated in the rescueof the axonopathy caused by overexpression of protein tau (Spittaels etal., J. Biol Chem 2000; 275: 41340-41349.). This interesting findingagain opens various experimental possibilities for functionalcomplementation as demonstrated below.

Combined, these results constitute the needed evidence that theheterologous expression of human protein tau in yeast results inphosphorylation at the specified phospho-epitopes that are also found inbrain of Alzheimer patients. Hence, heterologous expression of humanprotein tau in yeast provides a system that yields or recapitulates someor all of the specified phospho-epitopes that are hypothesised to beimportant in the formation of neurofibrillary tangles, and thereby causethe tau-opathy and contribute to the pathology of neurodegenerativediseases like Alzheimer's disease. In addition, some of the resultsprovide indications for completely novel experimental systems to addressthe signal-transduction cascade pathways that are responsible for andlead to the hyper-phosphorylation of protein tau at those specifiedepitopes.

Example 3 Expression of Human Protein Tau in Saccharomyces cerevisiae:Heterologous Expressed Human Protein Tau is Less Phosphorylated in YeastStrains Overexpressing the Protein Phosphatase PP2A

To illustrate that heterologous expressed protein tau is a substrate forprotein phosphataes in yeast, we monitored tau-phosphorylation in awild-type strain and a wild-type strain with overexpression of PPH21,one of the yeast genes encoding the phosphatase PP2A.

Results: As shown in FIG. 3, overexpression of PPH21 and hence increasedPP2A activity, enhanced immunoreactivity of protein-tau for the antibodyTAU-1 which recognizes non-phosphorylated epitopes.

In agreement to what has been suggested based on in vitro data withmammalian extracts, our data demonstrate that protein-tau isdephosphorylated in vivo when the activity of PP2A is increased inyeast. This surprising result demonstrates again that yeast can functionas model study and to identify novel components involved indephosphorylation of protein tau.

Example 4 Expression of Human Protein Tau in Saccharomyces Cerevisiae:Functional Complementation: Yeast Kinases Recognise the samePhosphorylation Sites in Protein Tau as their Human Homologues

To find out if yeast kinases can be functionally complemented by andrecognise the same phospho-epitopes in vivo as their homologous humankinases, we overexpressed the human GSK-3β in the mds1Δ strain (lackingone of the yeast genes encoding a kinase homologous to the humantau-kinase GSK-3β) and monitored tau-phosphorylation in crude extractsmaking use of monoclonal anitbody AD2.

Results: As shown in FIG. 4, the immunoreactivity of human protein tauto AD2 is restored in the mds1Δ strain when the human GSK-3β isco-expressed. This demonstrates that both the endogenous and theheterologous expressed GSK-3β kinases phosphorylate the phospho-epitopesof AD2, i.e. Ser-396 and Ser404.

These results provide evidence for functional complementation betweenyeast and human signal transduction components and confirm that theseevolutionary diverged proteins maintained a high degree of substratespecificity.

Example 5 Expression of Human Protein Tau in Saccharomyces Cerevisiae:Yeast as a Model to Elucidate the Signal-Transduction Pathways thatControl (Hyper)Phosphorylation of Protein Tau

To study the signal transduction cascades that controltau-phosphorylation, we monitored phosphorylation of human protein tauin strains deficient in different kinase activities, i.e PKA, Sch9 andYak1. We and others have previously demontrated that these kinasesoperate in interconnecting nutrient-induced pathways in yeast (TheveleinJ. M., Yeast, 1994; 10: 1753-1790; Crauwels et al., Microbiol., 1997;143: 2627-2637; Hartley, A. D. et al., Genetics, 1994; 136: 465-474). Nodata are available that their human homologues, resp. PKA, PKB/Akt1 andDYRK-1, directly phosphorylate protein tau in vivo but several researchgroups reported their involvement in tau-(hyper)phosphorylation andneurodegeneration based on in vitro results (Bilingsley M. L. andKincaid, R. L. Biochem. J. 1997;323: 577-591; Woods, Y. L. et al.,Biochem. J. 2001; 355: 609-615).

Results: The strong immunoreactivity with the monoclonal antibody Tau-1observed upon Western blot analysis of crude extracts obtained from thedifferent tau-expressing kinase deletion strains confirms that thesekinases have a role in the phosphorylation process of tau in yeast (FIG.5).

Of particular interest is the difference in phosphorylation of wild-typetau and mutant tau- P301L in the strain without PKA activity (tpk1Δtpk2Δ tpk3Δ msn2Δ msn4Δ) and the strain expressing only one of the threecatalytic subunits of PKA, i.e. TPK3 (tpk1Δ tpk2Δ TPK3 msn2Δ msn4Δ). Thestrong immunoreactivity observed in the former, is completely abolishedby the presence of Tpk3 for tau-P301L but only partially reduced forwild-type tau. This confirms the interference of tau mutations in thephosphorylation process (cfr example 1). In addition, the limitedreduction of immunoreactivity to Tau-1 of wild-type tau in the TPK3strain illustrates that Tpk3 can only mediate partial phosphorylation ofSer-199 and Ser-202 and that full PKA activity is required for completephophorylation. These findings demonstrate that engineered yeast strainscan be used as a model to study the signal transduction cascade thatcontrols tau phosphorylation.

Conclusions: These results provide the first in vivo data for acontribution of PKA, Sch9 (PKB/Akt) and Yak1 (DYRK-1) in the control oftau-phosphorylation. Whether this implies a direct phosphorylation, i.e.that these kinases recognise tau as a substrate, cannot be concluded butthis can be addressed by further epistasis analysis. Given that allthree yeast kinases can be functionally complemented by their respectivemammalian homologues as we and others have reported (Geyskens, I. Etal., Nato Sci. Ser., 2000; 316: 117-126; Yan, B. et al., Yeast, 2001; 18(S1): S273; Zhang, Z. et al., Mol. Biol. Cell., 2001; 12: 699-710), theresults demonstrate that the engineered tau-expressing yeast strains canserve as In vivo models to unravel the signal transduction pathways andto identify novel components that control tau-phosphorylation.

Example 6 Expression of Human Protein Tau in Saccharomyces Cerevisiae:Heterologous Expression of Human Protein Tau Affects PhysiologicalProcesses in Yeast Allowing High Throughput Screening Procedures to beDeveloped

To investigate how that heterologous over-expression of human proteintau in yeast affected specified physiological processes, we monitoredthe formation of pseudo-hyphae and sensitivity to the antimitoticfungicide benomyl. Under limiting growth conditions, such as growth onpoor nitrogen sources, certain yeast strains undergo dimorphictransition to an apparent filamentous growth form, referred to aspseudohyphal differentiation. This not only requires changes in thebudding pattern and cell adhesion but also cell elongation and hencealteration of the microfilament cytoskeleton (Gancedo, J. M., Femsmicrobiol. Rev., 2001;25:107-123; Winsor B and Schiebel E, Yeast, 1997;13:399434). Benomyl, like nocodazole, is a microtubule destabilisingagent and inhibitor of microtubule polymerisation (Hoyt M. A., Cell,1991; 6:507-517; Carminat J. L., Stearns, T., J. Cell Biololgy, 1997;138: 629-641).

Results pseudohyphal growth: The wild-type yeast strain Σ1278b wastransformed either with wild-type tau, mutant tau-P301L or an emptyplasmid and grown for 4 days under conditions of nitrogen limitation toinduce pseudohyphal differentiation. As seen in FIG. 6A, expression ofhuman wild-type protein tau interfered considerably or even preventedpseudohyphae formation under these conditions, while mutant tau-P301Linhibited pseudohyphae formation to a lesser extend than wild-type tau.

Results benomyl sensitivity: When yeast BY4741strains were grown on YPDmedium supplemented with benomyl, differences in sensitivity could beobserved dependent on the deletion present and on whether wild-type tauor mutant tau-P301L was expressed. As illustrated in the growth curvesin FIG. 6B, the wild type strain is less resistant to 40 μg/ml benomylwhen wild-type tau is expressed. In the mds1Δ strain (lacking one of theyeast genes encoding a kinase homologous to the human tau-kinaseGSK-3β), both wild-type and mutant tau-P301L improved resistance whereasin the pho85Δ strain (deficient for a kinase which is homologous to thesecond known tau-kinase, i.e. cdk5), increased resistance was observedonly with expression of mutant tau-P301L. With the pho85Δ strain, thiseffect was also obtained when cells are plated on benomyl-containing YPDmedium. Already with concentrations as low as 10 μg/ml benomyl, improvedgrowth was observed for the pho85A strain transformed with mutanttau-P301L (FIG. 6C). These results demonstrate that also in yeast theheterologous expressed tau interferes with microtubule function.

Conclusion: These results demonstrate clearly that heterologousover-expression of human protein tau affects specified measurablephysiological processes in yeast. Since the differences in phenotypesdepend on the expression of wild-type tau or mutant tau-P301L and on theprotein kinase activities present in the strains, the data constituteevidence supporting the use of engineered yeast for high throughputscreenings for compound affecting various tau-properties includingtau-phosphorylation and tau-microtubule-interaction.

Example 7 Expression of Human Protein Tau in Saccharomyces Cerevisiae:Solubility-Assay of Heterologous Expressed Human Protein Tau in Yeast

To analyse the solubility of heterologous expressed human protein tau,sequential extractions of yeast cells (grown in SD-ura till an OD600 of2) with high salt reassembly buffer (RAB), detergent containingradioimmunoprecipitation assay buffer (RIPA) and 70% formic acid (FA)was performed as described. For detection, we used the monoclonalantibody Tau-5.

Results: As shown in FIG. 7, wild type tau and mutant tau P301L arepredominantly present as insoluble protein in the yeast strains tested.Tau proteins could only be partially solubilsed in RAB and RIPA buffer.With 70% formic acid, a minor fraction remained insoluble in thewild-type BY4741 and the isogenic mds1Δ strain but not in the pho85Δmutant. In the latter, wild-type tau and mutant tau P301L weredetectable in the supernatant but not in the pellet.

It can be concluded that tau is predominantly found as insoluble proteinin yeast indicating that also in this organism tau forms aggregates andthat the aggregation is depending on the phosphorylation status ofprotein tau. Hence, yeast can be validated as model to study selfassembly of tau and to elucidate the formation of paired helicalfilaments. In addition, such a yeast model offers the opportunity to usetau to drive the aggregation of a selectable marker and as such developa high throughput screening strategy for components that specificallyinterfere with tau aggregation.

Whereas aggregation of Tau is rather time-laborious to monitor, fusionproteins of Tau with enzymatically active proteins appear to show thesame phosphorylation-dependent aggregation, and in this case aggregationcoincides with the removal of the corresponding enzymatic activity fromthe cell. This is illustrated by the fusion protein of Tau and thekanamycine-resistance gene product, which is soluble in the mds1Δ strain(lacking one of the yeast genes encoding a kinase homologous to thehuman tau-kinase GSK-3β), but present in an aggregated form in the wildtype background. Concomitant with this observation, strains expressingthe kanr-Tau fusion in a wild type background are kanamycine sensitive,whereas strains expressing kanr-Tau fusions in the mds1 background arekanamycine-resistant. Any compound inhibiting Mds1 preventsphosphorylation of the kanr-Tau fusion and hence results inkanamycine-resistance. Hence compounds which induce growth of kanr-Taufusion expressing wild type cells on kanamycine-containing media arecandidate-inhibitors of Mds1. Replacement of Mds1 with its humanhomologue GSK3β renders this screening assay specific for inhibitors ofhuman GSK3β.

Compounds that cause solubilisation of the kanr-Tau fusion via amechanism different from GSK3 inhibition will also yield a positiveread-out in this assay. These GSK3-independent ‘Tau solubilisers’ arelikely to represent interesting new classes of (therapeutic) compoundsactive against tau-o-pathies. Since monitoring of Tau expression duringgrowth revealed that Tau expression occurred only after 5-10 hours ofgrowth, compounds that reverse Tau aggregation as well compounds thatprevent Tau aggregation can be identified depending on the time ofadministration of the compound to the yeast cultures. Cultures can begrown in microtiter plates and administration of compounds andmonitoring of growth (optical density at 600 nm) can be fully automated,allowing the scaling up of the method and the screening of chemicallibraries.

The principle of the method described can also be applied to strainsexpressing fusions of Tau with any other reporter, such as URA3, whichhas the advantage that functional Ura3 results in growth on mediumwithout uracil, but in toxicity on FOA-containing media allowing acomplementary approach and screening.

References to the Application

-   Billingsley M L, Kincaid R L., Biochem J 1997;323:577-591-   Brion J P,. Eur. Neurol., 1998, 40: 130-140.-   Delacourte A Dement Geriatr Cogn Disord, 199, 10: 75-79-   DEWACHTER I., MOECHARS D., VAN DORPE J., SPITTAELS K., TESSEUR I.,    VAN DEN HAUTE C. and-   VAN LEUVEN F., Experimental Gerontology, 35, 831-841, 2000-   Hardy J., Duff K, Hardy K G, Perez-Tur J, Hutton M, Nat Neurosci,    1998, 1: 355-358-   Heutink P., Hum. Molec. Genet., 2000, 9: 979-986.-   Mandelkow E M and Mandelkow E, 1998, Trends Cell-Biol., 8: 425-427.-   Sambrook, J. and Russel D W, 2001 “Molecular Cloning: A laboratory    manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y.-   Selkoe, D. ( 2000 ) Neurol Clin., 18: 903-922-   Spittaels, K., Van den Haute, C., Van Dorpe, J., Vandezande, K.,    Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A.,    Loos, R., Van Leuven, F. , Am. J. Pathol., (1999) 155: 2153-2165-   Spittaels K, Van den Haute C, Van Dorpe J, Geerts H, Mercken M,    Bruynseels K, Lasrado R, Vandezande K, Laenen I, Boon T, Van Lint J,    Vandenheede J, Moechars D, Loos R, Van Leuven F., J. Biol Chem 2000,    275: 41340-41349.-   Tesseur I., Van Dorpe J., Spittaels K., Van den Haute C., Moechars.    D, Van Leuven F., Am. J. Pathol., 2000, 156, 951-964-   Tesseur, I., Van Dorpe, J., Bruynseels, K., Bronfman, F., Sciot, R.,    Van Lommel, A. and Van Leuven, F., Am. J. Pathol., 2000, 157,    1495-1510.-   Van Dorpe, J., Smeijers, L., Dewachter, I., Nuynens, D., Spittaels,    K., Van Den Haute, C., Mercken, M., Moechars, D., Laenen, I.,    Kuiperi, C., Bruynseels, K., Tesseur, I., Loos, R., Vanderstichele,    H., Checler, F., Sciot, R. and Van Leuven, F. Am. J. Pathol. ,2000,    157, 1283-1298.

VAN LEUVEN F., Progress in Neurobiology, 61, 305-312, 2000 TABLE 1Oligonucleotide sequences used to delete specific genes in S. cerevisiaeORF name Oligosequence 5′-3′ MDS1 CACGGCAACACTAATCACGCAACGC forwardTAGAACTCGCGCAGGAGATTGTACT GAGAGTGCAC TCTCCCATTATTCTTGCCTGGGCTCC reversedCTCCGGTGCTATCACTGTGCGGTAT TTCACACCG PHO85 ATGTCTTCTTCTTCACAGTATGTAGTforward TTTCTAGTCAAGTATCATTGGAAAGT AAAGAACTAGAAATGATGAATACTAACAAGATTGTACTGAGAGTGCAC TTATGAAGCGTGGTGGTAGTACTCTG reversedCAAACCAAGGGTGATGCAGAGCCTG CTTGGCGCTCAGCCTCATATCCGGATTAACTGTGCGGTATTTCACACCG SCH9 ATGATGAATTTTTTTACATCAAAATC forwardGTCGAATCAGGATACTGGATTTAGCT CTCAACACCAACATCCAAATGGACAGAAAGATTGTACTGAGAGTGCAC TCATATTTCGAATCTTCCACTGACAA reversedATTCGTCATCCATGTGTTGGTCGCCG TCGAAGTCCATCTGCGAATGGCTGTTGTCTGTGCGGTATTTCACACCG YAK1 AGTTAAGAAAAGTCAGACAAAATAA forwardCGATTTATTCTTCGAAGATTGTACT GAGAGTGCAC AGCAATCATGAACTCATCCAATAAT reversedAACGACTCGTCCAGCCTGTGCGGTA TTTCACACCGSequences in bold indicate complementarity to the pRS plasmid.

1-74. (cancelled)
 75. An engineered microbial yeast, comprising anintroduced nucleotide sequence or an allelic variant, minigene, asynthetic gene or a homologue thereof coding for tau:
 76. The engineeredmicrobial yeast of claim 75, comprising an introduced mammaliannucleotide sequence or an allelic variant, minigene or a homologuethereof coding for a tau.
 77. The engineered microbial yeast of claim75, wherein tau is a wild type protein tau, a protein tau isoform, aprotein tau mutant or a functional homologue thereof.
 78. The engineeredmicrobial yeast of claim 75, wherein said introduced mammaliannucleotide sequence is correctly integrated to allow the heterologousexpression of tau.
 79. The engineered microbial yeast of claim 75,wherein endogenous yeast protein kinases exhibit phosphorylation and anendogenous yeast protein phosphastase modulates phosphorylation of saidtau.
 80. The engineered microbial yeast of claim 75, which furthercomprises an introduced DNA sequence comprising a promoter, correctlyintegrated to direct the expression of a yeast kinase or yeastphosphatase that modulates phosphorylation of said tau.
 81. Theengineered microbial yeast of claim 75, which further comprises anintroduced DNA sequence encoding a human or mammalian kinase orphosphatase that modulates the phosphorylation of said tau, undercontrol of a promoter sequence.
 82. The engineered microbial yeast ofclaim 75, which further comprises one or more of the following: anintroduced DNA sequence comprising a promoter, correctly integrated todirect the expression of a yeast glycogen synthase kinase-3 beta or ahomologous yeast protein, that modulates the phosphorylation of saidtau. an introduced DNA sequence encoding a glycogen synthasekinase-3beta or a homologous yeast protein, that modulates thephosphorylation of said tau, under control of a promoter sequence. anintroduced DNA sequence comprising a promoter, correctly integrated todirect the expression of a yeast cdk5 or a homologous protein, thatmodulates the phosphorylation of said tau. an introduced DNA sequenceencoding a cdk5 or a homologous protein, that modulates thephosphorylation of said tau under control of a promoter sequence. 83.The engineered microbial yeast of claim 82, wherein said microbial yeastwhen cultured exhibits phosphorylation or hyperphosphorylation of saidtau.
 84. The engineered microbial yeast of the claim 82, which comprisesat least one further kinase that can modulate the phosphorylation oftau, selected from a non-limiting group of kinases consisting of anAGC-kinase, a mitogen activated kinase, a glycogen synthase kinase, acdk5 kinase, a mitogen -activated kinase, a cdc2 kinase, another brainproline-directed kinase, a MEK kinase, a RAS and a GEF kinase or ahomologous proteins with a protein kinase function or at least onefurther phosphatase that can modulate the phosphorylation of tau,selected from a non-limiting group of proteins consisting of PP1phosphatases, PP2A or PP2A-like phosphatases, PP2B phosphatases, PP2Cphosphatases or other proteins with a protein phosphatase function. 85.The engineered microbial yeast of claim 75, characterised in that saidyeast has been modulated to have modified yeast signal-transductioncascade pathways.
 86. The engineered microbial yeast of claim 85,wherein said modulation results in a deletion mutant of an endogenousyeast kinase or phosphatase.
 87. The engineered microbial yeast of claim86, characterized in that said modulation corresponds to the deletionobtained in the MDS1Δ, the PHO85Δ, the SCH9Δ or the YAK1 Δ deletionmutants.
 88. The engineered microbial yeast of claim 75, wherein saidtau is expressed using a constitutive promotor.
 89. The engineeredmicrobial yeast of claim 75, wherein tau is expressed using an induciblepromotor.
 90. The engineered microbial yeast of claim 75, wherein thenucleotide sequence encoding tau is fused to a secretion signal.
 91. Theengineered microbial yeast of claim 75, wherein said nucleotide sequenceor allelic variant, minigene, synthetic gene or homologue thereof codingfor tau is coupled in-frame to a reporter protein and is correctlyintegrated to allow the expression or overexpression of a reporterprotein-tau fusion protein.
 92. The engineered microbial yeast of claim75, wherein tau drives the precipitation of the tau-reporter fusionprotein and thereby inhibits or changes the biological function of thereporter protein.
 93. The engineered microbial yeast of claim 75,wherein said engineered microbial yeast is transformed with a yeastexpression cDNA library whereby said cDNA is derived from human and/ormammalian tissues, including brain tissue and whereby the effect of saidcDNA on tau function and/or tau phosphorylation and biochemistry can bemonitored.
 94. The engineered microbial yeast of claim 75, whereby saidyeast is further modified either genetically or chemically to facilitatethe uptake of agents, compounds or chemical signals.
 95. The engineeredmicrobial yeast of any of the claim 75, which is of the order of theSaccharomycetales.
 96. A method of screening a plurality of agents,compounds or chemical signals that directly or indirectly affect tauphosporylation, comprising a) culturing, growing or suspending anengineered microbial yeast, comprising an introduced nucleotide sequenceor an allelic variant, minigene, a synthetic gene or a homologue thereofcoding for tau in an appropriate medium, b) adding said agents, compoundor chemical signal to said engineered microbial yeast or its medium, c)measuring the extent to which said tau phosphorylation and/or functionis affected, d) identifying those agents, compounds or chemical signalsfor which an effect on tau phosphorylation and/or function in the yeastis observed.
 97. The method of claim 96 wherein said engineeredmicrobial yeast has been modified either genetically or chemically tofacilitate the uptake of agents, compounds or chemical signals thatdirectly or indirectly affect tau phosphorylation and/or function. 98.The method of claim 98 wherein said engineered microbial yeast has beenmodulated to have modified yeast signal-transduction cascade pathways.99. The method of claim 96, wherein said modulation results in adeletion mutant of an endogenous yeast kinase or phosphatase.
 100. Themethod of claim 96, further comprising comparing the effect of saidagents, compounds or chemical signals on said engineered microbialyeast, with the effect thereof on engineered microbial yeast that isdeficient in the expression of said tau or which is deficient in theexpression of a protein kinase that modulates phosphorylation of saidtau.
 101. The method of claims 22, further comprising comparing theeffect of said agents, compounds or chemical signals on said engineeredmicrobial yeast, with the effect thereof on engineered microbial yeastthat is deficient in the expression of said tau or which is deficient inthe expression of a protein phosphatase that modulates thephosphorylation of said tau.
 102. The method of claim 96, wherein stepc) comprises using an antibody, preferably a monoclonal antibody, or afab thereof capable of binding selectively to phosporylated orunphosphorylated tau.
 103. The method according to claim 101 forscreening a plurality of agents, compounds or chemical signals forbinding to and modulation of the activity of any of the protein kinasesof the group consisting of an AGC-kinase, a mitogen activated kinase, aglycogen synthase kinase, a cdk5 kinase, a mitogen -activated kinase, acdc2 kinase, another brain proline-directed kinase, a MEK kinase, a RASand a GEF kinase or a homologous protein with a protein kinase function.104. The method according to claim 101, for screening a plurality ofagents, compounds or chemical signals for binding to and modulation ofthe activity of any of the protein phosphatases of the group consistingof PP1 phosphatases, PP2A or PP2A-like phosphatases, PP2B phosphatases,PP2C phosphatases or other proteins with a protein phosphatase function.105. A method of screening a plurality of agents, compounds or chemicalsignals for activity that directly or indirectly modulates tauphosporylation, function or solubility comprising a) culturing, growingor suspending an engineered microbial yeast, comprising an introducednucleotide sequence or an allelic variant, minigene, a synthetic gene ora homologue thereof coding for tau in an appropriate medium, b) addingsaid agents, compounds or chemical signals to said engineered microbialyeast or its medium, c) detecting or quantitating specified biologicalor cell morphogenic process in the yeast d) identifying those agents,compounds or chemical signals for which an effect on said specifiedbiological or cell morphogenic process in the yeast is observed. 106.The method of claim 105, wherein said specified biological or cellmorphogenic processes comprise formation of mitotic bundles, formationof pseudo-hyphen, formation of scar-sites, cell-size, cell metabolism,cell survival or cell growth in defined conditions.
 107. A method foridentifying the structure-function relation of phosphorylated mutant tauproteins, wherein the method involves a) culturing, growing orsuspending an engineered microbial yeast, comprising an introducednucleotide sequence or an allelic variant, minigene, a synthetic gene ora homologue thereof coding for tau, characterised in that it expresses amutant tau protein and a protein kinase, in an appropriate medium, b)culturing, growing or suspending an engineered microbial yeast,comprising an introduced nucleotide sequence or an allelic variant,minigene, a synthetic gene or a homologue thereof coding for tau,characterised in that it expresses said mutant tau protein but isdefective in a gene coding for a protein kinase, in an appropriatemedium, c) comparing specified biological processes of said kinaseeffective and kinase defective engineered microbial yeast.
 108. A methodfor identifying the structure-function relation of said phosphorylatedmutant tau proteins, wherein the method involves a) culturing, growingor suspending an engineered microbial yeast, comprising an introducednucleotide sequence or an allelic variant, minigene, a synthetic gene ora homologue thereof coding for tau, characterised in that they express amutant tau protein and a protein phosphatase, in an appropriate medium,b) culturing, growing or suspending an engineered microbial yeast,comprising an introduced nucleotide sequence or an allelic variant,minigene, a synthetic gene or a homologue thereof coding for tau,characterised in that it expresses said mutant tau protein but isdefective in a gene coding for a protein phosphatase, in an appropriatemedium, c) comparing specified biological processes of said phosphataseeffective and phosphatase defective engineered microbial yeast.
 109. Amethod for the production of a pharmaceutical composition comprising themethod of claim 96 and furthermore mixing the compound identified or aderivative or homologue thereof with a pharmaceutically acceptablecarrier.
 110. A purified protein identified using the screening methodof claim 96, which modulates protein tau phosporylation, function orsolubility.
 111. A pharmaceutical composition comprising a protein ofclaim 110, in conjunction with a pharmaceutical acceptable excipient.112. A method for the production of a pharmaceutical compositioncomprising the method of claim 105 and furthermore mixing the compoundidentified or a derivative or homologue thereof with a pharmaceuticallyacceptable carrier.
 113. A purified protein identified using thescreening method of claim 105, which modulates protein tauphosporylation, function or solubility.
 114. A pharmaceuticalcomposition comprising a protein of claim 113, in conjunction with apharmaceutical acceptable excipient.
 115. A method for the production ofa pharmaceutical composition comprising the method of claim 107 andfurthermore mixing the compound identified or a derivative or homologuethereof with a pharmaceutically acceptable carrier.
 116. A purifiedprotein identified using the screening method of claim 107, whichmodulates protein tau phosporylation, function or solubility.
 117. Apharmaceutical composition comprising a protein of claim 116, inconjunction with a pharmaceutical acceptable excipient.
 118. A methodfor the production of a pharmaceutical composition comprising the methodof claim 108 and furthermore mixing the compound identified or aderivative or homologue thereof with a pharmaceutically acceptablecarrier.
 119. A purified protein identified using the screening methodof claim 108, which modulates protein tau phosporylation, function orsolubility.
 120. A pharmaceutical composition comprising a protein ofclaim 119, in conjunction with a pharmaceutical acceptable excipient.